1. Field of the Invention
The present invention relates to an optical system for a holographic microscope.
2. Description of the Related Art
Popescu et al. (Optics Letters, v. 31, pages 775-777, 2006 (Diffraction phase microscopy for quantifying cell structure and dynamics)) have developed diffraction phase microscopy, which is shown in FIG. 1, as a technique for quantitative phase imaging of biological structures. The method combines the principles of common path interferometry and single-shot phase imaging and is characterized by subnanometer path-length stability and millisecond-scale acquisition time. The potential of the technique for quantifying nanoscale motions in live cells is demonstrated by experiments on red blood cells.
An experimental setup is illustrated in FIG. 1. VPS means a virtual source point, RL is a relay lens, G is a grating, IP is an image plane, L1,2 are lenses (f1,2, respective focal distances), SF means a spatial filter, and CCD is used as a detector.
In this Letter by Popescu et al., they introduce diffraction phase microscopy (DPM) as a novel quantitative phase imaging technique. DPM uses the common path geometry and provides quantitative phase images that are inherently stable to the level of the subnanometer optical path length and at an acquisition speed limited only by the detector.
The second harmonic (λ=532 nm) radiation of a Nd:YAG laser was used as illumination for an inverted microscope (Axiovert 35, Carl Zeiss, Inc.), which produces a magnified image of the sample at the output port. The microscope image appears to be illuminated by the virtual source point (VPS). The relay lens (RL) was used to collimate the light originating at the VPS and replicate the microscope image at the image plane (IP).
A phase grating G is placed at this image plane (IP), which generates multiple diffraction orders containing full spatial information about the image. They isolate the zeroth and first diffraction orders to be used as sample and reference fields, respectively, similar to typical Mach-Zender interferometry. To accomplish this, a standard spatial filtering lens system L1-L2 is used to select the two diffraction orders and generate the final interferogram at the CCD plane.
The zeroth order beam is low-pass filtered by using the spatial filter (SF) positioned in the Fourier plane of L1, such that at the CCD plane it approaches a uniform field. The spatial filter allows passing the entire frequency content of the first diffraction order beam and blocks all the other orders.
The first order beam is thus the imaging field and the zeroth order beam plays the role of the reference field. The two beams traverse the same optical components, i.e., they propagate along a common optical path, thus significantly reducing the longitudinal phase noise. The direction of the spatial modulation was chosen at an angle of 45° with respect to the x and y axes of the CCD, such that the total field at the CCD plane has the form described in the following equation (1).E(x,y)=|EO|ei[φ0β(x+y)]|E1(x,y)|eiφ(x,y)  (1)
In Eq. (1), |EO| and |E1| are the amplitudes, and φ0 and φ are the phases of the orders of diffraction 0, 1, respectively, while β represents the spatial frequency shift induced by the grating to the zeroth order. Note that, as a consequence of the central ordinate theorem, the reference field is proportional to the spatial average of the microscope image field described below.
                                                                    E              0                                            ⁢                      ⅇ                          ⅈϕ              0                                      ∝                              1            A                    ⁢                      ∫                                                                                                E                    1                                    ⁡                                      (                                          x                      ,                      y                                        )                                                                              ⁢                              ⅇ                                  ⅈϕ                  ⁡                                      (                                          x                      ,                      y                                        )                                                              ⁢                              ⅆ                x                            ⁢                              ⅆ                y                                                                        (        2        )            where A is the total image area. The spatial average of an image field has been successfully used before as a stable reference for extracting spatially resolved phase information. The interferogram is spatially high-pass filtered to isolate the cross term, |EO∥E1(x,y)|cos [φ(x,y)−β(x+y)−φ0]. For the transparent objects of interest here, E1(x,y) is expected to have a weak spatial dependence. The spatially resolved quantitative phase image associated with the sample is retrieved from a single CCD recording via a spatial Hilbert transform.
The efficiency of the grating G depends on the angle of use. On the image plane IP, where the grating is placed illustrated in FIG. 1, the signal beam has wide angular contents which carry the information of interest. Therefore, the grating will modify the angular contents of the signal; thus, may change the image of the object under investigation.